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Quantitative analysis of ERG expression and its splice isoforms in formalin fixed, paraffin embedded prostate cancer samples: Association with seminal vesicle invasion and biochemical recurrence

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Quantitative analysis of ERG expression and its splice isoforms in formalin fixed paraffin embedded prostate cancer samples: Association with seminal vesicle invasion and biochemical recurrence.

Rachel M. Hagen, Ph.D1; Patricia Adamo, Ph.D1; Saima Karamat, M.Sc2; Jon Oxley, M.D. FRCPath2; Jonathan J Aning M.D.3; David Gillatt, M.D3; Raj Persad, M.D3; Michael R. Ladomery, Ph.D1*; Anthony Rhodes, Ph.D1, 4*#

1. Faculty of Health & Life Sciences, University of the West of England, Bristol, UK. 2. Department of Cellular Histopathology, North Bristol NHS Trust, Bristol, UK. 3. North Bristol NHS Trust, Bristol, UK.

4. Department of Pathology, University of Malaya Medical Centre, Kuala Lumpur, Malaysia

* Joint senior authors

Page proofs, correspondence, and requests for reprints should be directed to:-

Prof. Anthony Rhodes, Dept of Pathology, Faculty of Medicine, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia. Email: Anthony.Rhodes@uwe.ac.uk, Anthony.Rhodes@ummc.edu.my, Tel: (603) 7949 2064. Fax: (603) 7955 6845

Source of Funding: The authors would like to thank the funding bodies for this work. RMH is supported by the Bristol Urological Institute, North Bristol NHS Trust and the University of the West of England. PA was supported by the Rotary Club of Bristol and the Bristol Urological Institute.

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DISCLOSURE

The authors declare that there are no conflicts of interests.

ABSTRACT

Objectives: The proto-oncogene ETS-related gene (ERG) is consistently overexpressed in prostate cancer. Alternatively spliced isoforms of ERG have variable biological activities; inclusion of exon 11 (72bp) is associated with aggressiveness and progression of disease. Exon 10 (81bp) has also been shown to be alternatively spliced. Within this study we assess whether total ERG protein, mRNA and ERG splice isoform mRNA expression is altered as prostate cancer progresses.

Methods: Detection of the TMPRSS2:ERG fusion was done using direct methods (RT-PCR and FISH) and indirect methods for ERG mRNA and protein expression using qPCR and immunohistochemistry, respectively. A linear equation method was used to quantitatively determine relative proportions of ERG variants (ERG72/∆72, ERG81/∆81) for each sample.

Results: ERG mRNA and protein expression is increased in patients with advanced prostate cancer, with a trend for up regulation in advanced versus localised disease, with higher levels of ERG expression significantly associated with seminal vesicle invasion (Stage pT3b) and biochemical recurrence. Genes involved in cell migration and invasiveness (Matrix metalloproteinase 7, osteopontin and septin 9) are increased in prostate cancers that overexpress ERG. In addition, there is a clear indication of increased retention of exons 10 and 11 in prostate cancer.

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INTRODUCTION

The transcription factor ERG (ETS Related Gene) is overexpressed in 60-80% of prostate cancer cases [1-3] and is often attributed to a fusion between the promoter of the TMPRSS2 gene and the coding region of ERG [4,5]. In benign prostate ERG expression levels are low and are not regulated by androgens; however in prostate cancer ERG levels are significantly higher especially if fused to the TMPRSS2 promoter which is under the control of androgens. Expression analysis of prostate cancers reveals a wide array of genes potentially regulated by ERG to include genes involved in cell proliferation such as septin 9 (SEPT9) and metastatic pathways such as matrix metalloproteinases (e.g. MMP 3/7/9), osteopontin (OPN) and E-cadherin [3, 7, 8].

However, the clinical importance of ERG overexpression and the presence of the TMPRSS2-ERG fusion in prostate cancer is still unclear as there are reports of a positive, negative and zero correlation with development and aggressiveness of prostate cancer.[9-11].

Added complexity of fusion transcripts arise due to alternative splicing. Wild type ERG consists of 17 exons and expresses multiple splice isoforms [12, 13]. A significant finding has been that TMPRSS2-ERG and wild-type ERG variants exhibit differing biological properties [13, 14]. A common alternative splicing event within the central activation exon domain (CAE) of ERG is inclusion/skipping of a 72 bp exon (herein referred to as exon 11). Recent studies in vitro suggest that the inclusion of the 72bp exon (exon 11) results in increased cell proliferation and a more oncogenic phenotype [13]. Exon 10 (81bp) can also be alternatively spliced [13].

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progresses. If so the analysis of ERG expression and its splice variants in routine clinical samples of prostate cancer rather than just the genomic fusion, as detected by FISH, may be of value in determining the prognosis.

MATERIALS AND METHODS

Tissue samples

This study utilised tissue samples from fifty-three patients diagnosed between 2000-2009 with clinically localised hormone naïve adenocarcinoma of the prostate (Table 1) (NRES No. 09/H0102/48). Cases were selected based on the availability of cases for review and large enough tumour foci for sampling, excluding any clinically insignificant cases. Whole prostatectomy samples were fixed in neutral buffered saline for 24 hours before processing to paraffin wax and embedding in Mega Tissue Cassettes (Tissue-Tek®, Netherlands). For the study, areas of benign and invasive prostate carcinoma were identified, cored and re-embedded in regular sized cassettes (Tissue-Tek®,). In order to validate the use of RT-PCR to detect the fusion, a total of 20 of the 53 cases were randomly selected for both FISH analysis and RT-PCR. Subsequently, RT-PCR and qPCR for ERG variants was performed on 53 cases.

Fluorescent in situ hybridisation (FISH) analysis.

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Immunohistochemistry

Each of 40 patient samples were stained using two antibodies to ERG (EP111, Dako, UK and EPR3864; Epitomics, USA), in addition to double staining with both ERG EP111 and high molecular weight cytokeratin (34BE12, Dako, UK). All immunohistochemistry, to include antigen retrieval was performed on a BondMax™ instrument (Leica Microsystems, UK), using Bond™ Epitope Retrieval Solution. Nuclear ERG staining was visualised using the Bond™ Polymer Refine Detection system, with a diaminobenzidine chromogen. Cytoplasmic

staining for high molecular weight cytokeratin staining was visualised using the Bond™ Polymer Refine Red Detection. Immunohistochemical expression of ERG was assessed as described previously, using a four tier grading system in which the intensity of nuclear staining is recorded as; negative, weakly positive, moderately positive and strongly positive [17]. The specificity of the ERG antibodies was verified by western blot analysis. In addition, we compared the staining pattern for both the EP111 clone and the previously validated EPR3864 clone [17], both giving identical results on all cases. The EP111 clone was used for comparative analysis with the other variables, due to reduced background. Cases with

invasive prostatic adenocarcinoma showing strong nuclear positivity by both clones served as positive controls. In addition, occasional endothelial cells and lymphocytes stained positively for ERG and served as internal controls. Omission of the primary antibodies, served as negative controls.

RNA isolation

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overnight. RNA yields were determined by A260 measurement using a Nanodrop spectrophotometer (Thermo Fischer Scientific, USA). Reverse transcription was performed using 500ng of RNA and M-MLV Reverse Transcriptase (Promega, UK) as per manufacturers specifications.

RT- PCR analysis

Primers to detect TMPRSS2:ERG fusion were as designed by Tomlins et al [7]. TMPRSS2:ERG forward 5.-TAGGCGCGAGCTAAGCAGGAG-3’ and TMPRSS2:ERG reverse 5’-GTAGGCACACTCAAACAACGACTGG-3’. PCR was carried out using GoTaq

Hot Start Polymerase (Promega) per manufacturers recommendations.

Quantitative realtime PCR

Quantitative realtime PCR was performed using 2x Sybr green master mix (Roche, UK) and primers at 300 nmol concentration on TaqMan7300 Sequence detection System (Applied Biosystems). Primers were designed to span at least one exon boundary using the Primer Express 2.0 software (Applied biosystems, UK) and were purchased from Sigma-Genosys (Haverhill, UK) using standard qPCR cycling conditions (Table 2). Fold changes in expression were calculated by using a standard curve method [18]. Data were normalised to the corresponding β-actin value for each sample.

Quantitative analysis of ERG variants using LEM-PCR

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Statistics

Data from experiments are presented as mean +/- standard error mean (SEM), with numbers of replicates stated in figure legends. Statistical significance between variables was tested using,the paired two-tailed student’s t-test,a Kruskal-Wallis test and the Pearson Chi-Squared Test. Biochemical recurrence after radical prostatectomy was defined as a PSA value greater than or equal to 0.2ng/ml, with a second confirmatory level of PSA of >0.2 ng/ml (33).

RESULTS

ERG mRNA expression is increased in TMPRSS2-ERG fusion positive samples.

We analysed TMPRSS2-ERG gene fusion in cancer samples using both RT-PCR and FISH in an initial 20 cases selected at random to determine concordance between the techniques (Figure 1). The RT-PCR reaction detected one, two or no TMPRSS2-ERG fusion variants (Figure 1A). RT-PCR and FISH results were highly concordant (18/20), with RT-PCR giving an additional two cases as TMPRSS2-ERG positive compared with FISH. Using RT-PCR on all 53 cases within this study we identified 58% (31/53) of samples to have a TMPRSS2-ERG fusion event. ERG mRNA expression determined using real-time PCR was significantly increased in TMPRSS2-ERG fusion positive cancer samples compared with fusion negative samples, p<0.01(Figure 1C).

Analysis of ERG mRNA and protein expression in prostate cancer cases

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staining by immunohistochemistry for ERG had significantly higher levels of ERG mRNA expression (17.99±5.49), as determined by qPCR, than cases with none or low immunohistochemical staining for ERG (4.59±1.94, p=0.019; Figure 2B). ERG mRNA expression was significantly up regulated in both localised (Stage T2, p=0.000416) and advanced cancer (Stages T3A, p=0.00397 and T3B, p=0.04120) cases when compared with benign prostate tissue (Figure 2C). In addition, ERG was significantly up regulated in Stage 3 cancer compared to Stage T2 (p=0.009512).

Quantitative analysis of ERG target gene expression in prostate cancer FFPE samples that either have low or high ERG gene expression

Samples were designated as having high levels of ERG (ERG_high) if they had a 2-fold increase in ERG compared with benign tissues. On average the ERG_high subset had a 16-fold increase in ERG mRNA expression compared with the ERG_low subset, p=0.0022 (Figure 3A). Increased mRNA expression for Matrix metalloproteinase 7 (MMP7) p=0.3483 osteopontin (OPN)p=0,0468 and septin 9 (SEPT9)o=0.00697 mRNA expression was seen in prostate tumours with high ERG mRNA expression compared with low ERG mRNA expressing tumours (Figure 3B).

Quantitative analysis of ERG expression is associated with seminal vesicle invasion and biochemical recurrence.

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Relative ERG CAE splice isoform expression between benign and prostate tumours

In benign tissue the relative proportions of ERG72/∆72 and ERG81/∆81 were roughly equal (Table 4). However, the percentage of total ERG mRNA that was accounted for by ERG∆72 (exon 11 skipped) and ERG∆81(exon 10 skipped) was significantly decreased in both T3A and T3B advanced cancer cases compared with benign tissue and there was a trend for decreased ERG∆72 and ERG∆81 in both Stage T3a and T3b advanced cancer cases when compared with T2 localised cancer (Figure 4 and Table 4).

DISCUSSION

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transcripts as a result of inclusion of premature stop codons [20, 21]. Additionally, if there is TMPRSS2-ERG fusion but androgen signalling is absent or disrupted there will be none or little ERG expression in prostate cancer cells [22-24]. As such TMPRSS2-ERG fusion status may therefore not always reflect the levels of ERG present. Thus instead of detecting a TMPRSS2-ERG fusion the accurate measurement of ERG expression may be of more prognostic relevance. Here we show that up-regulation of ERG results in increased expression of genes involved in cell proliferation (septin 9), and metastases (metalloproteinase 7, and osteopontin) (3, 7, 8), and that high levels of ERG expression are significantly associated with seminal vesicle invasion and biochemical recurrence.

Our study also highlights the potential of immunohistochemistry as a high throughput assay for detection of overexpression of ERG in clinical cases. Immunohistochemistry has become increasingly utilised as a surrogate marker for the TMPRSS2-ERG fusion status. a [17, 25, 26]. It is important to note that whilst TMPRSS2 is the most common fusion partner for ERG, ERG can also be rearranged and fused with the SLC45A3 and NDRG1 genes. These alternative fusion partners can account for approximately 5% of ERG overexpressing prostate cancers [27-29]. As such using a TMPRSS2-ERG FISH probe in isolation could result in missing a number of prostate cancers that would have significantly elevated levels of ERG. Like FISH, immunohistochemistry allows for marker expression to be localised in relation to tumour morphology. However, in comparison to FISH, it is relatively inexpensive, technically less demanding and readily assessed under the light microscope.

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and 3’ splice sites and intron retention [32]. However, as the retention of the 72bp exon in

ERG increases cell proliferation and invasion in vitro it is highly possible that the changes in relative proportions of ERG variants may significantly contribute to the progression of prostate cancer. Future studies will focus on addressing the significance of these ERG splice variants in larger cohorts and whether these splice variants may predispose an individual to advanced prostate cancer. If found to be clinically relevant, the technology described readily lends itself to the testing of ERG and its variants on smaller needle biopsies, and the possibility of providing guidance to clinicians on the need for radical treatment.

ACKNOWLEDGEMENTS

The authors would also like to thank Jess Broadhurst (University of Bristol) and Martin Figgitt (University of Bristol) for technical assistance with the FISH assay and visualisation. We also thank Dako, UK for provision of the ERG EP111 clone.

REFERENCES

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3. Iljin K, Wolf M, Edgren H, et al. TMPRSS2 fusions with oncogenic ETS factors in

prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming. Cancer Res. 2006;66:10242-10246.

4. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005;310:644-648.

5. Tomlins SA, Laxman B, Dhanasekaran SM, et al. Distinct classes of chromosomal

rearrangements create oncogenic ETS gene fusions in prostate cancer. Nature 2007;448:595-599.

6. Oikawa T. ETS transcription factors: Possible targets for cancer therapy. Cancer.Sci. 2004;95:626-33.

7. Flajollet S, Tian TV, Flourens A, et al. Abnormal expression of the ERG transcription factor in prostate cancer cells activates osteopontin. Mol.Cancer.Res. 2011;9:914-924.

8. Gupta S, Iljin K, Sara H, et al. FZD4 as a mediator of ERG oncogene-induced WNT signaling and epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res. 2010;70:6735-6745.

9. Cerveira, N, Ribeiro FR, Peixoto A, et al. TMPRSS2-ERG gene fusion causing ERG overexpression precedes chromosome copy number changes in prostate carcinomas and paired HGPIN lesions. Neoplasia 2006;8:826-832.

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11. St John J, Powell K, Conley-Lacomb MK, et al. TMPRSS2-ERG fusion gene expression in prostate tumor cells and its clinical and biological significance in prostate cancer

progression. J.Cancer.Sci.Ther. 2012;4:94-101.

12. Carrere S, Verger A, Flourens A, et al. Erg proteins, transcription factors of the ets family, form homo, heterodimers and ternary complexes via two distinct domains. Oncogene 1998;16:3261-3268.

13. Wang J, Cai Y, Ren C, et al. Expression of variant TMPRSS2/ERG fusion messenger RNAs is associated with aggressive prostate cancer. Cancer Res. 2006;66:8347-8351.

14. Wang J, Cai Y, Yu W, et al. Pleiotropic biological activities of alternatively spliced TMPRSS2/ERG fusion gene transcripts. Cancer Res. 2008;68:8516-8524.

15. Hu Y, Dobi A, Sreenath T, et al. Delineation of TMPRSS2-ERG splice variants in prostate cancer. Clin.Cancer Res. 2008;14:4719-4725.

16. Chin SF, Daigo Y, Huang HE, et al. A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples. Mol.Pathol. 2003;56:275-279.

17. Park K, Tomlins SA, Mudaliar KM, et al. Antibody-based detection of ERG rearrangement-positive prostate cancer. Neoplasia 2010;12:590-598.

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19. Virtue S, Dale M, Sethi JK, et al. LEM-PCR: A method for determining relative transcript isoform proportions using real-time PCR without a standard curve. Genome 2010;53:637-642.

20. Soller MJ, Isaksson M, Elfving P, et al. Confirmation of the high frequency of the TMPRSS2/ERG fusion gene in prostate cancer. Genes Chromosomes Cancer 2006;45:717-729.

21. Clark J, Merson S, Jhavar S, et al. Diversity of TMPRSS2-ERG fusion transcripts in the human prostate. Oncogene 2007;26:2667-2673.

22. Hermans KG, van Marion R, van Dekken H, et al. TMPRSS2:ERG fusion by

translocation or interstitial deletion is highly relevant in androgen-dependent prostate cancer, but is bypassed in late-stage androgen receptor-negative prostate cancer. Cancer Res.

2006;66:10658-10663.

23. Saramaki OR, Harjula AE, Martikainen PM, et al. TMPRSS2:ERG fusion identifies a subgroup of prostate cancers with a favorable prognosis. Clin.Cancer Res. 2008;14:3395-3400.

24. Linja MJ, Savinainen KJ, Saramaki OR, et al. Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res. 2001;61:3550-3555.

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26. Tomlins SA, Palanisamy N, Siddiqui J, et al. Antibody-based detection of ERG rearrangements in prostate core biopsies, including diagnostically challenging cases: ERG staining in prostate core biopsies. Arch.Pathol.Lab.Med. 2012;136:935-946.

27. Han B, Mehra R, Dhanasekaran SM, et al. A fluorescence in situ hybridization screen for E26 transformation-specific aberrations: Identification of DDX5-ETV4 fusion protein in prostate cancer. Cancer Res. 2008;68:7629-7637.

28. Esgueva R, Perner S, LaFargue CJ, et al. Prevalence of TMPRSS2-ERG and SLC45A3-ERG gene fusions in a large prostatectomy cohort. Mod.Pathol. 2010;23:539-546.

29. Pflueger D, Rickman DS, Sboner A, et al. N-myc downstream regulated gene 1 (NDRG1) is fused to ERG in prostate cancer. Neoplasia 2009;11:804-811.

30. Jhavar S, Reid A, Clark J, et al. Detection of TMPRSS2-ERG translocations in human prostate cancer by expression profiling using GeneChip human exon 1.0 ST arrays.

J.Mol.Diagn. 2008;10:50-57.

31. Iwamoto M, Higuchi Y, Koyama E, et al. Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development. J.Cell Biol. 2000;150:27-40.

32. Kim E, Goren A, and Ast G. Insights into the connection between cancer and alternative splicing. Trends Genet. 2008;24:7-10.

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Recommendations for a Standard in the Reporting of Surgical Outcomes. The Journal of Urology 2007; 177: 540-545.

TABLE AND FIGURE LEGENDS

Figure 1.

A) RT-PCR was performed on cDNA generated from cancerous regions of prostate from 20 cases using primers directed against exon1 of TMPRSS2 and exon 4 of ERG to detect the ERG fusion. PCR products were sequenced to confirm the identity of TMPRSS2-ERG fusion variants. B) Representative image of a fusion positive and a fusion negative case obtained using a triple colour breakapart TMPRSS2-ERG FISH probe. C) Comparison of total ERG mRNA expression in TMPRSS2-ERG positive (n=26) and TMPRSS2-ERG negative (n=14) FFPE cases as determined by qPCR (**p<0.01).

Figure 2.

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Figure 3.

qPCR analysis of ERG target gene expression in prostate cancers with high and low values of ERG mRNA expression. ERG_high was defined as a 2-fold or greater increase in ERG compared with benign tissues. A) Total ERG; on average the ERG_high subset (n=24) had a 16-fold increase in ERG mRNA expression compared with the ERG_low subset (n=16), **p=0.0022, B) ERG-high cancers show increased expression of mRNA for the target genes; Matrix metalloproteinase 7 (MMP7 (p=0.3483), osteopontin (OPN) *p=0.0468 and septin (SEPT9) **p=0.00697.

Figure 4.

Relative proportions of ERG variants plus/minus 72bp exon 11of ERG and plus/minus 81bp exon 10 of ERG expressed as percentages of the total ERG present in benign (BN, n=12), localised (pT2, n=21) and advanced (pT3A, n=19 and pT3B, n=13) prostate cancer samples as determined using the LEM-PCR method.

Table 4.

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Fusion Positive Fusion Negative

A.

B.

0 2 4 6 8 10 12 14 T ota l E RG (ERG /B -A CT IN)

C.

**

TMPRSS2:ERG GAPDH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sample No.

A. B.

1 2

3 4 5 ERG

1 2

4 5 ERG

TMPRSS2 ERG

A.

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B.

A U (E RG /B -A CT IN)

**

**

$

A.

0 5 10 15 20 25

No IHC Staining (n=21) ERG IHC Staining (n=19) A U (E RG /B -A CT IN)

C.

H and E

ERG/Cytokeratin

ERG

Localised

Cancer

Advanced

Cancer

0 2 4 6 8 10 12 14 16

Benign T2 T3A T3B

***

Cancer: Pathological Stage

*

No/Low ERG (IHC) (n=21)

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A U (E RG /B -A CT IN)

**

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

MMP7 OPN SEPT9

**

*

A U (G en e/B -A CT IN)

A.

B.

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[image:22.540.90.420.45.693.2]

Figure 4

T o ta l E RG (%) T o ta l E RG (%)

Figure 4

0

20

40

60

80

100

120

Benign T2 T3A T3B

ERG∆72

ERG72

Cancer: Pathological Stage

0 20 40 60 80 100 120

Benign T2 T3A T3B

ERG∆81

ERG81

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[image:23.595.62.483.176.565.2]

Table 1. Clinical parameters for patients used in this study

Variable Localised prostate cancer Advanced prostate cancer

No. of cases 21 32

Median PSA level ± SD, ng/mL

5.34 ± 2.25 12.80 ± 5.39

Gleason score cat. No.

3+3 = 6 13 8

3+4 = 7

4+4= 8+

6

0

22

4

Pathologic Stage, No.

pT2 21 0

pT3A

pT3B

0

0

19

13

Comment [A1]:

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[image:25.595.67.536.134.490.2]

Table 2: PCR primer sequences used.

Forward (5’-3’) Reverse (5’-3’)

β-Actin GCATGGAGTCCTGTGGCATCC A

ATCCTGTCGGCAATGCCAGGGT A

Total ERG (exon 16)

CATCTCCTTCCACAGTGCCCA CTGGATTTGCAAGGCGGCTAC

ERG∆72bp AGAAACACAGATTTACCATAT GAGC

ACCGGTCCAGGCTGATCTC

ERG 72bp exon

CCTGAAGCTACGCAAAGAATT ACA

ACCGGTCCAGGCTGATCTC

ERG 81bp TCTCCACGGTTAATGCATGC GAAAATAAAAGCTGCACCCCCT ERG∆81bp TCACATCTCCACTACCTCAGA

GA

TTGGGAAAATAAAAGCTGCAC

MMP7 GAACGCTGGACGGATGGT CATACCCAAAGAATGGCCAAGT

SEPT9 GGAGCGCATCCCCAAGA CGGACGCCTTTCTCCTCAA

OPN TGGCTAAACCCTGACCCATCT TCATTGGTTTCTTCAGAGGACA

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[image:26.595.59.533.129.621.2]

Table 3. Clinico-pathological parameters for patients designated with either low or high ERG mRNA expression.

Variable ERG_low ERG_high

No. of cases 25 28

ERG mRNA expression± SD, 1.03 ± 0.10 16.72 ± 5.34

Median PSA level ± SD, ng/mL

7.70 ± 1.52 7.82 ± 1.12

Gleason score cat. No.

3+3 = 6 11 10

3+4 = 7

4+4= 8+

13

1

15

3

Pathologic Stage, No.

pT2 12 9

pT3A

pT3B

10

3

9

10

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[image:28.595.82.516.181.355.2]

Table 4. Relative proportions of ERG with/without the 72bp or 81bp exon in clinical prostate samples

Average ± SEM p-value (ERG72 vs. ERG∆72)

ERG72 (%) ERG∆72 (%) BN pT2 pT3A pT3B

BN 51.71 ± 8.64 48.29 ± 8.09

0.55 0.0012 0.00009 0.00054

pT2 72.45 ± 7.51 27.64 ± 7.55

pT3A 86.47 ± 6.23 13.53 ± 6.24

pT3B 81.82 ± 3.61 18.18 ± 3.61

Average ± SEM p-value (ERG81 vs. ERG∆81)

ERG81 (%) ERG∆81 (%) BN pT2 pT3A pT3B

BN 57.52 ± 4.87 42.48 ± 7.12

0.68 0.008 0.015 0.013

pT2 76.37 ± 2.53 23.63 ± 2.75

pT3A 94.55 ± 3.27 5.45 ± 12.87

Figure

Figure 4 Figure 4
Table 1. Clinical parameters for patients used in this study
Table 2: PCR primer sequences used.
Table 3. Clinico-pathological parameters for patients designated with either low or high ERG mRNA expression
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References

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